The Signal-Seeker™ line of produts have been developed to allow simple analysis of key regulatory protein modifications by specialists and non-specialists alike. The comprehensive Signal-Seeker™ kits provide an affinity bead system to isolate and enrich modified proteins from any given cell or tissue lysate. The enriched protein population is then analyzed by standard western blot procedures using a primary antibody to the target protein.
Product Uses Include
Validation Data: SUMOylation 2/3 Detection Kit White Paper
The SUMOylation 2/3 kit contains the following components:
|Lysis and protein quantitation step||IP and pre-clear step||Wash step||Elution step||Western step|
BlastR™ Lysis Buffer
BlastR™ Dilution Buffer
Protease Inhibitor Cocktail
De-SUMOylation inhibitor Cocktail
Precision Red™ Protein Assay Reagent
SUMOylation 2/3 Affinity Beads
IP Control Beads
BlastR™ Wash Buffer
Bead Elution Buffer
Chemiluminescent Reagent A
Chemiluminescent Reagent B
There are many applications for these kits, here we describe an interesting example:
Application 1: Investigate significant SUMOylation 2/3 events
Immunoprecipitation using the Signal-Seeker™ SUMOylation 2/3 Detection Kit
Denatured cell lysates were prepared as previously reported1 from HeLa cells ("HS43": Heat Shock treated 42°C for 10 min, and "CT37": untreated) and HeLa siRNA SUMO knockdown ("KDS2"). 1mg of lysate was used for the immunoprecipitation (IP) of SUMOylated 2/3 proteins. Western blots of immunoprecipitated proteins were developed using anti-SUMO-2/3 antibody (Cytoskeleton cat# ASM23) (A) or anti-Ubc9 antibody (B). The level of SUMO-2/3 conjugates in heat shock treated cells is higher than control, and shRNA SUMO-2 knock-down reduced the level of SUMOylated 2/3 modified proteins. Chemical conjugation of SUMO-2/3 antibody (11G2) to the affinity bead matrix prevents heavy and light chain leaching. Unconjugated free SUMO is also captured by the SUMO-2/3 affinity beads.
(B) Unmodified Ubc9 is visible near 18kDa. High molecular-weight band indicates that Ubc9 is SUMOylated by a single SUMO-2/3 protein. Ubc9 has previously been reported to be a target for SUMOylation1,2.
1. Barysch S. et al. 2014. Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies. Nat Protoc. 9(4):896-909
2. Becker J. et al. 2013. Detecting endogenous SUMO targets in mammalian cells and tissues. Nature Struc. & Mol. Biol. 20, 525-531.
• Pharmacological investigation of SUMOylating and de-SUMOylating enzymes involved in regulation of target proteins.
• Investigate SUMOylation under a variety of different growth factors or drug treatments.
• Examine the interaction of SUMOylated target proteins with its downstream effectors.
• Examine crosstalk between SUMOylation 2/3 and other PTMs for target proteins.
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Signal-Seeker™ Phosphotyrosine Detection Kit (Cat. # BK160)
Signal-Seeker™ Ubiquitination Detection Kit (Cat. # BK161)
Signal-Seeker™: BlastR™ Rapid Lysate Prep Kit (Cat. # BLR01)
Signal-Seeker™ SUMOylation 2/3 Affinity Beads (Cat.# ASM24-beads)
Signal-Seeker™: PTMtrue™ SUMOylation 2/3 Antibody (Cat.# ASM23)
|Juncker, Meredith et al.||ISG15 attenuates post-translational modifications of mitofusins and congression of damaged mitochondria in Ataxia Telangiectasia cells||Biochimica et Biophysica Acta - Molecular Basis of Disease||2021||ISSN 1879-260X|
|Kim, Catherine et al.||SUMOylation of mitofusins: A potential mechanism for perinuclear mitochondrial congression in cells treated with mitochondrial stressors||Biochimica et Biophysica Acta - Molecular Basis of Disease||2021||ISSN 1879-260X|
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